PROJECT SUMMARY Chemical weapons were used in world wars, and more recently in Iraq and Syria. Acrolein, a volatile aldehyde, used during World War I caused severe problems in eye, skin and lungs in addition to many casualties. At present, there are no countermeasures available that can prevent, impede or arrest corneal damage and loss of vision caused by acrolein poisoning. Since acrolein is a vital precursor for variety of commercial goods, over 500,000 tons of acrolein is produced and transported annually in America, which pose a real threat of its use as a chemical weapon by the terrorists for mass causalities. It is our central hypothesis is that acrolein contact to eye depletes glutathione and augments various cytokines entry into the corneal stroma resulting in corneal damage and vision loss. This proposal aims to characterize molecular mechanisms involved in acrolein- exposed loss of corneal function and test the efficacy and safety of Glutamate Cysteine Ligase Catalytic subunit (GCLC; a rate-limiting enzyme in the synthesis of GSH) gene therapy given to cornea via our reported nanoparticlebased gene transfer method to mitigate acrolein-induced toxicity and vision loss in vivo using a preclinical rabbit model. To the best of our knowledge, no scientific data is currently available on mechanisms mediating acrolein toxicity and gene-based therapy. Aim-1 will delineate molecular and cellular mechanisms of acrolein intoxication to the eyes by studying changes in the glutathione content, reactive oxidative stress, keratocyte death, and transforming growth factor ?1 in corneal tissues, and cytokines level in tears after acrolein contact to eye in a rabbit model. These factors are known to cause ocular irritation, inflammation, corneal scarring and corneal neovascularization in vivo. Aim-2 will test the hypothesis that nanoparticle-based GCLC gene therapy enhancing the glutathione redox system will counteract deleterious effects of acrolein in the cornea by stimulating the synthesis of GSH and increasing the antioxidant index in vivo in a rabbit model. To accomplish proposed research, we have selected a rabbit model that recapitulates clinical traits of acrolein toxicity observed in human patients. Rabbit eyes will be exposed to acrolein and ocular health in live rabbits will be monitored with clinical slit-lamp and confocal microscopes. The changes at the cellular and molecular levels in tears and corneal tissues will be analyzed using commercial assays such as TUNEL, GSH, ROS etc., immunoblotting, immunofluorescence, qPCR etc. techniques. Our team is comprised of corneal researchers, physician and veterinary ophthalmologists with extensive experience, skills, tools, and joint publications, and is most suitable for this translational research. Successful completion of this project will lead better understanding of molecular mechanisms and pathways modulated by acrolein in the eye, and will provide necessary in vivo data that will lead development of gene-based nanomedicine approaches to treat acrolein-induced ocular toxicity and blindness. The nanoparticle-based gene transfer method that will be used has showed transgene expression for two weeks after one topical application in rabbit cornea in vivo.